This revised proposal is aimed at defining molecular mechanisms of intercompartmental protein transport from both the secretory and endocytic pathways to the lysosome. Correct regulation of protein transport in the secretory and endocytic pathways is essential for normal function in all eukaryotic cells. Defective intracellular protein traffic occurs in such diverse human diseases as breast cancer and Alzheimer's syndrome. The lysosome is one organelle that shows particular dispositions to protein traffic errors as nearly 40 disorders in lysosomal storage exist and protein turnover in lysosomes is central to treating certain neurodegenerative diseases. Understanding the molecular details of how eukaryotic cells specifically transport proteins to lysosomes will have broad relevance to fundamental cell biology. These fundamental issues are best answered with a diverse experimental design. In this research, the yeast, Saccharomyces cerevisiae, will be used as a model eukaryotic organism because molecular, genetic, and biochemical approaches are readily integrated for protein transport investigations to its lysosome- like organelle, the vacuole. Integrating genetics and biochemistry has lead to a more thorough elucidation of intracellular protein transport mechanisms during the early secretory pathway. This work, however, specifically focuses on movement of proteins from the newly-discovered prevacuolar endosomal compartment to the lysosome/vacuole, which is poorly understood in both mammals and yeast. The greatest area of uncertainty in this process concerns whether transport carrier vesicles shuttle cargo between these organelles or late endosomes "mature" into lysosomes. To prove or disprove these models, experiments are designed to establish the hypothesis that specific carrier vesicles function to transport proteins from the prevacuolar compartment to the lysosome/vacuole. Using a combination of in vitro reconstitution assays and molecular genetics, proteins that function in vesicle formation, vesicle transport, or vesicle consumption will be identified and studied. Specifically, (1) cytosolic proteins will be purified and functionally characterized using intercompartmental protein transport assays in permeabilized cells; (2) develop new in vitro assays using subcellular fractions to measure stage- specific events in transport from the prevacuolar compartment to the vacuole; (3) the identity of carrier vesicles that operate to transport protein cargo from the prevacuolar compartment to the vacuole will be established; and (4) the role a selected subset of vacuolar protein sorting (vps) defective yeast mutants has in endosome to lysosome protein traffic will be determined.